ERK Ligands and Polynucleotides Encoding ERK Ligands

ABSTRACT

The invention relates to kinase inhibitor ligands and polyligands. In particular, the invention relates to ligands and polyligands that modulate ERK activity. The ligands and polyligands are utilized as research tools or as therapeutics. The invention includes linkage of the ligands and polyligands to a cellular localization signal, epitope tag and/or a reporter. The invention also includes polynucleotides encoding the ligands and polyligands.

This application claims benefit of priority to provisional application60/865,589 filed 13 Nov. 2006.

FIELD OF INVENTION

The invention relates to mammalian kinase ligands, substrates andmodulators. In particular, the invention relates to polypeptides,polypeptide compositions and polynucleotides that encode polypeptidesthat are ligands, substrates, and/or modulators of ERK. The inventionalso relates to polyligands that are homopolyligands orheteropolyligands that modulate ERK activity. The invention also relatesto ligands and polyligands tethered to a subcellular location.

This application has subject matter related to application Ser. No.10/724,532 (now U.S. Pat. No. 7,071,295), 10/682,764 (US2004/0185556,PCT/US2004/013517, WO2005/040336), Ser. No. 11/233,246, andUS20040572011P (WO2005116231). Each of these patents and applications ishereby incorporated by reference.

BACKGROUND OF THE INVENTION

The ability to modulate protein activities has long been the hallmark ofsmall molecule drug discovery and development, and the success of thistraditional therapeutic approach is unquestioned. However, the numberand nature of small molecule drug targets are more limiting than wouldbe ideal and have less target specificity and more off-target sideeffects that will likely make for significant commercial and regulatorychallenges in the years ahead. A newer technology for inhibiting proteinactivity that has received acceptance is siRNA-mediated gene silencing.The mechanism for siRNA inhibition is post-transcriptional andpre-translational. It has the advantage of being relatively selectivefor target RNA sequences but, like small molecules, suffers fromoff-target side effects.

Kinases are enzymes that catalyze the addition of phosphate to amolecule. The addition of phosphate by a kinase is calledphosphorylation. When the kinase substrate is a protein molecule, theamino acids commonly phosphorylated are serine, threonine and tyrosine.Phosphatases are enzymes that remove phosphate from a molecule. Theremoval of phosphate is called dephosphorylation. Kinases andphosphatases often represent competing forces within a cell to transmit,attenuate, or otherwise modulate cellular signals and cellular controlmechanisms. Kinases and phosphatases have both overlapping and uniquenatural substrates. Cellular signals and control mechanisms, asregulated by kinases, phosphatases, and their natural substrates are atarget of research tool design and drug design.

Mammalian mitogen-activated protein kinase (MAPK) andextracellular-signal-regulated kinase (ERK) are the same enzyme, hereinreferred to as ERK. ERK has two isoforms, both of which canphosphorylate serine and threonine residues in protein or peptidesubstrates. Use of the term ERK herein encompasses both ERK isoforms.Many cellular substrates of ERK have been identified. Furthermore,polypeptides have been used to examine ERK substrate specificity. Whilepolypeptides and variants thereof have been studied as individualsubstrates or ligands, mixed ligands linked together as polyligands thatmodulate ERK activity have not been demonstrated before this invention.An aspect of the invention is to provide novel, modular, inhibitors ofERK activity by modifying one or more natural substrates by truncationand/or by amino acid substitution. A further aspect of the invention isthe subcellular localization of an ERK inhibitor, ligand, or polyligandby linking to a subcellular localization signal. Examples of ERKsubstrates and/or regulators include those described in the followingreferences: Adams, et al. 2000 J Neurochem 75:2277-87, Arnaud, et al.2004 J Immunol 173:3962-71, Chung, et al. 1997 Mol Cell Biol 17:6508-16,Clark-Lewis, et al. 1991 J Biol Chem 266:15180-4, Eymin, et al. 2006Cell Cycle 5:759-65, Fantz, et al. 2001 J Biol Chem 276:27256-65,Garcia, et al. 2002 Embo J 21:5151-63, Gille, et al. 1995 Embo J14:951-62, Haycock, et al. 1992 Proc Natl Acad Sci USA 89:2365-9,Hedges, et al. 2000 Am J Physiol Cell Physiol 278:C718-26, Hindley, etal. 2002 J Cell Sci 115:1575-81, Howell, et al. 1991 Mol Cell Biol11:568-72, Ishibe, et al. 2004 Mol Cell 16:257-67, Jacobs, et al. 1999Genes Dev 13:163-75, Jacque, et al. 1998 Embo J 17:2607-18, Kelemen, etal. 2002 J Biol Chem 277:8741-8, Kolch 2000 Biochem J 351 Pt 2:289-305,Lefebvre, et al. 2002 J Cell Biol 157:603-13, Lin, et al. 1999 J BiolChem 274:15971-4, Matallanas, et al. 2006 Mol Cell Biol 26:100-16,Matsuura, et al. 2005 Biochemistry 44:12546-53, Matter, et al. 2002Nature 420:691-5, Missero, et al. 2000 Mol Cell Biol 20:2783-93, Morton,et al. 2004 FEBS Lett 572:177-83, Pandey, et al. 2005 Mol Cell Biol25:10695-710, Sanghera, et al. 1990 FEBS Lett 273:223-6, Schaeffer, etal. 1999 Mol Cell Biol 19:2435-44, Songyang, et al. 1996 Mol Cell Biol16:6486-93, Soond, et al. 2005 J Cell Sci 118:2371-80, Tenet, et al.2003 Development 130:5169-77, Veeranna, et al. 1998 J Neurosci18:4008-21, Xu, et al. 2001 Mol Cell Biol 21:2981-90, Zhang, et al. 2001J Biol Chem 276:14572-80, and MAP Kinase Substrate Peptide Catalog#2-125 Lot #23369 (Upstate, Lake Placid, N.Y.).

Design and synthesis of polypeptide ligands that modulatecalcium/calmodulin-dependent protein kinase and that localize to thecardiac sarco(endo)plasmic reticulum was performed by Ji et al. (J BiolChem (2003) 278:25063-71). Ji et al. accomplished this by generatingexpression constructs that localized calcium/calmodulin-dependentprotein kinase inhibitory polypeptide ligands to the sarcoplasmicreticulum by fusing a sarcoplasmic reticulum localization signal derivedfrom phospholamban to a polypeptide ligand. See also U.S. Pat. No.7,071,295.

DETAILED DESCRIPTION OF POLYPEPTIDE AND POLYNUCLEOTIDE SEQUENCES

SEQ ID NOS:1-8 are example polyligands and polynucleotides encodingthem.

Specifically, the ERK polyligand of SEQ ID NO:1 is encoded by SEQ IDNO:2, SEQ ID NO:3, and by SEQ ID NO:4, wherein the codons have beenoptimized for mammalian expression. SEQ ID NO:3 and SEQ ID NO:4 includedifferent alternatives of predetermined flanking restriction sites.Furthermore, SEQ ID NO:4 utilizes alternative codons for mammalianexpression. A vector map of a vector containing SEQ ID NO:4 is shown inFIG. 12 (labeled ERK decoy). SEQ ID NO:1 is an embodiment of apolyligand of the structure A-S1-B-S2-C-53-D-S4-E-S5-F, wherein A is SEQID NO:91, B is SEQ ID NO:97, C is SEQ ID NO:28, D is SEQ ID NO:29, E isSEQ ID NO:30, and F is SEQ ID NO:31, wherein Xaa is alanine, and whereinS1 is a spacer of the amino acid sequence AA, and S2 is a spacer ofamino acid sequence AAAA, S3 is a spacer of the amino acid sequenceGAGA, S4 is a spacer of the amino acid sequence GGGG, and S5 is a spacerof the amino acid sequence AGAG. A polyligand of structureA-S1-B-S2-C-S3-D-S4-E-S5-F is also called herein a heteropolyligand,shown generically in FIG. 4D.

SEQ ID NO:5 is an embodiment of a polyligand of the structureX-Y-S2-Z-S3-A-S4-B-S6-C-S5-D-S7-E-S8-F, wherein X is SEQ ID NO:32, Y isSEQ ID NO:98, Z is SEQ ID NO:33, A is SEQ ID NO:34, B is SEQ ID NO:35, Cis SEQ ID NO:100, D is SEQ ID NO:36, E is SEQ ID NO:37, and F is SEQ IDNO:107, wherein Xaa is alanine, and wherein S2 is a spacer of amino acidsequence AAAA, S3 is a spacer of the amino acid sequence GAGA, S4 is aspacer of the amino acid sequence GGGG, S6 is a spacer of the amino acidsequence AGPGAEF, S5 is a spacer of the amino acid sequence AGAG, S7 isa spacer of the amino acid sequence AAGG, and S8 is a spacer of theamino acid sequence GGAA. The ERK polyligand of SEQ ID NO:5 is encodedby SEQ ID NO:6, SEQ ID NO:7 and by SEQ ID NO:8, wherein the codons havebeen optimized for mammalian expression. SEQ ID NO:7 and SEQ ID NO:8include different alternatives of predetermined flanking restrictionsites. Furthermore, SEQ ID NO:8 utilizes alternative codons formammalian expression. A polyligand of structureX-Y-S2-Z-S3-A-S4-B-S6-C-S5-D-S7-E-S8-F is also called herein aheteropolyligand, shown generically in FIG. 4E.

SEQ ID NOS:9-27 are full length ERK protein substrates. These sequenceshave the following public database accession numbers: NP004032,NP001871, NP149129, NP001781, 075956, NP005220, NP536739, CAI17445,AAF65618, NP001006666, NP035353, NP062651, Q07666, AAL68976, NP644805,NP003174, Q15648, NP033411, and AAA42258. Each of the sequencesrepresented by these accession numbers is incorporated by referenceherein. In SEQ ID NOS:9-27, the positions of the amino acid(s)phosphorylatable by ERK are represented by Xaa. In wild-type proteins,Xaa is serine or threonine. In the ligands of the invention, Xaa is anyamino acid.

SEQ ID NOS:28-90 are peptide sequences including subsequences of SEQ IDNOS:9-27, which represent examples of kinase active site blocker peptideligand sequences where the location of the ERK phosphorylatable serineor threonine in the natural polypeptide is designated as Xaa.

SEQ ID NOS:91-108 are polypeptide inhibitors of ERK (see FIG. 15).Specifically, SEQ ID NOS:91-96 are ERK activation site blockers, and SEQID NOS:97-108 are ERK docking site blockers.

SEQ ID NOS:28-108 represent examples of monomeric polypeptide ligandsequences.

Amino acid sequences containing Xaa encompass polypeptides where Xaa isany amino acid.

DETAILED DESCRIPTION OF DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A-1C show examples of homopolymeric ligands without spacers.

FIGS. 2A-2C show examples of homopolymeric ligands with spacers.

FIGS. 3A-3E show examples of heteropolymeric ligands without spacers.

FIGS. 4A-4F show examples of heteropolymeric ligands with spacers. InFIG. 4E, the abbreviation, S, stands for SPACER.

FIGS. 5A-5G show examples of ligands and polymeric ligands linked to anoptional epitope tag.

FIGS. 6A-6G show examples of ligands and polymeric ligands linked to anoptional reporter.

FIGS. 7A-7G show examples of ligands and polymeric ligands linked to anoptional localization signal.

FIGS. 8A-8G show examples of ligands and polymeric ligands linked to anoptional localization signal and an optional epitope tag.

FIGS. 9A-9G show examples of gene constructs where ligands andpolyligands are linked to an optional localization signal, an optionalepitope tag, and an optional reporter.

FIGS. 10A-10D show examples of vectors containing ligand geneconstructs.

FIG. 11 shows an example of a sequential cloning process useful forcombinatorial synthesis of polyligands.

FIG. 12 shows a diagram of a vector for cell transformation.

FIG. 13 shows Cos7 cells transformed with the vector depicted in FIG.12, wherein the vector includes SEQ ID NO:4 which encodes the ERKpolyligand of SEQ ID NO:1. This figure demonstrates endoplasmicreticulum (ER) localization of an ERK polyligand: Cos7 cells weretransfected with vector containing an ER localization signal, a c-Mycepitope tag, and the ERK polyligand of SEQ ID NO:1 (ERK decoy). Panels Aand B depict Cos7 cells transfected with the ERK decoy while Panel Cdepicts a Cos7 cell transfected with a localization signal controlvector lacking an ERK polyligand. The cells in each panel were treatedwith a stain for the ER-resident protein calreticulin (red) as well asanti-c-Myc antibody staining specific to the c-Myc epitope tag (green).Panels A, B and C show concentrated protein expression to theendoplasmic reticulum as evidenced by the co-localization between boththe ERK decoy and localization control with the ER-resident proteincalreticulin (yellow).

FIG. 14 shows localized inhibition of ERK-mediated myelin basic proteinphosphorylation by the ERK polyligand of SEQ ID NO:1 (decoy). Aconstitutively-active form of the RasV 12 protein, a known activator ofMAPK signaling pathways, was used to activate ERK kinase in definedregions of Cos7 cells. Several fusion proteins as described byMatallanas et al. Mol Cell Biol. 2006 January; 26(1):100-116 (herebyincorporated by reference), were used to activate ERK kinase in specificsubcellular compartments. The constitutively-active RasV12 proteinpromoted cell-wide activation of ERK. The Lck-RasV 12 fusion proteinactivated ERK-protein associated with lipid rafts in or near the plasmamembrane. The M1-RasV12 fusion protein activated ERK in the endoplasmicreticulum. Lane 1: control. Lane 2: ERK activity in cells expressingactive Lck-RasV12 fusion protein. Lane 3: ERK activity in cellsco-expressing active Lck-RasV12 fusion protein, and ERK decoy protein(ER localization signal, a c-Myc epitope tag, and the ERK polyligand ofSEQ ID NO:1). Lane 4: ERK activity in cells co-expressing activeLck-RasV 12 fusion protein, and CAT fragment-containing ER localizationcontrol protein. Lane 5: ERK activity in cells expressing activeM1-RasV12 fusion protein. Lane 6: ERK activity in cells co-expressingactive M1-RasV12 fusion protein, and ERK decoy protein. Lane 7: ERKactivity in cells co-expressing active M1-RasV12 fusion protein, and CATfragment-containing ER localization control protein. Lane 8: ERKactivity cells expressing active RasV12 protein. Lane 9: ERK activity incells co-expressing active RasV12 fusion protein, and ERK decoy protein.Lane 10: ERK activity in cells co-expressing active RasV12 protein, andCAT fragment-containing ER localization control protein. This figurerepresents compartmentalized ERK activity in the plasma membrane (Lanes2-4), in the endoplasmic reticulum (Lanes 5-7), and cell wide (Lanes8-10) in Cos-7 cells. The bands on the gel represent varyingphosphorylation states of ERK substrate, myelin basic protein (MBP);darker bands represent higher levels of ERK activity. When SEQ ID NO:1was added to cells with ER-active ERK, ERK activity in the endoplasmicreticulum was reduced by approximately 60%. Again, Lane 1 is thecontrol. Lanes 2-4 show activated ERK at the plasma membrane. Lanes 5-7show activated ERK in the ER. Lanes 8-10 show activated ERK in theentire cell. Lanes 2, 5, & 8 show normal ERK activity. Lanes 3, 6, & 9show ERK activity with co-expressed SEQ ID NO:1 fusion protein. Lanes 4,7, & 10 show ERK activity with co-expressed control.

FIG. 15 shows a diagram of the ERK interaction sites of the differentcategories of ERK monomeric ligands including active site blockers,docking site blockers, and activation site blockers.

FIG. 16 shows nuclear localization of SEQ ID NO:1 fused to a nuclearlocalization signal and c-Myc epitope tag. Location was detected byimmunostaining for c-Myc (green).

FIG. 17 shows cytoplasmic localization of SEQ ID NO:1 fused to anuclear-exclusion localization signal and c-Myc epitope tag. Locationwas detected by immunostaining for c-Myc (green).

FIG. 18 shows inhibition of cell proliferation using ERK polyligands ofSEQ ID NO:1 and SEQ ID NO:5 as compared to siRNA and a small moleculeinhibitor. G418-resistant colony formation was assayed in NIH3T3 cellsusing siRNA specific for ERK1 or ERK2 isoforms; a small moleculeinhibitor; and pancellular (no localization signal) polyligands of SEQID NO:1 and SEQ ID NO:5. G418-resistant colony formation was assayed inNIH3T3 cells transfected with vector (C) (1 μg) plus: siRNAoligonucleotides for ERK isoform 1 (Si1) or ERK isoform 2 (Si2) (25 ng);or vectors encoding for SEQ ID NO:1 (Dy1) or SEQ ID NO:5 (Dy2) (1 μg);or treated with the MEK inhibitor UO126 (1 μM). Colonies were stainedand counted after 15 days in culture.

FIG. 19 shows inhibition of cell proliferation using localized ERKpolyligand SEQ ID NO:1 fused to different localization signals.G418-resistant colony formation was assayed in NIH3T3 cells usingpancellular SEQ ID NO:1 and SEQ ID NO:5, or SEQ ID NO:1 targeted toeither the cytoplasm (NXP, nuclear exclusion), nucleus (NLS), plasmamembrane (PLA), or endoplasmic reticulum (ER). G418-resistant colonyformation was assayed in NIH3T3 cells transfected with vector (C) (1 μg)plus constructs (1 μg each) encoding for SEQ ID NO:1 (Dy1), SEQ ID NO:5,(Dy2) or SEQ ID NO:1 targeted to: cytoplasm (NXP), nucleus (NLS), plasmamembrane (PLA), and endoplasmic reticulum (ER). Colonies were stainedand counted after 15 days in culture.

FIG. 20 shows inhibition of cell transformation with pancellular SEQ IDNO:1 (Dy1), pancellular SEQ ID NO:5 (Dy2), and ER-localized SEQ ID NO:1(ER1), and plasma membrane-localized SEQ ID NO:1 (PLA1) as compared tosiRNA against ERK isoform 2 (Si2). Transformed foci formation wasassayed in NIH3T3 cells transfected with H-ras V12 or v-Src (0.25 ng)plus constructs (1 μg each).

BRIEF DESCRIPTION OF THE INVENTION

The invention relates to polypeptide ligands and polyligands for ERK.Various embodiments of the ERK ligands and polyligands are representedin SEQ ID NOS:1-108. More specifically, the invention relates toligands, homopolyligands, and heteropolyligands that comprise any one ormore of SEQ ID NOS:28-108. Additionally, the invention relates toligands and polyligands comprising one or more subsequences of SEQ IDNOS:9-27 or any portion thereof. Furthermore, the invention relates topolyligands with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% and99% sequence identity to a polyligand comprising one or more of SEQ IDNOS:28-108 or any portion thereof. Furthermore, the invention relates topolyligands with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% and99% sequence identity to a polyligand comprising one or moresubsequences of SEQ ID NOS:9-27.

Polyligands, which can be homopolyligands or heteropolyligands, arechimeric ligands composed of two or more monomeric polypeptide ligands.An example of a monomeric ligand is the polypeptide represented by SEQID NO:38, wherein Xaa is any amino acid. SEQ ID NO:38 is a selectedsubsequence of wild-type full length SEQ ID NO:9, wherein the amino acidcorresponding to Xaa in the wild-type sequence is a serine or threoninephosphorylatable by ERK. An example of a homopolyligand is a polypeptidecomprising a dimer or multimer of SEQ ID NO:38, wherein Xaa is any aminoacid. An example of a heteropolyligand is a polypeptide comprising SEQID NO:28 and one or more of SEQ ID NOS:29-108, wherein Xaa is any aminoacid. There are numerous ways to combine SEQ ID NOS:28-108 intohomopolymeric or heteropolymeric ligands. Furthermore, there arenumerous ways to combine additional subsequences of SEQ ID NOS:9-27 witheach other and with SEQ ID NOS:28-108 to make polymeric ligands.

The polyligands of the invention optionally comprise spacer amino acidsbefore, after, or between monomers. SEQ ID NO:1 is an embodiment of apolyligand of the structure A-S1-B-S2-C-S3-D-S4-E-S5-F, wherein A is SEQID NO:91, B is SEQ ID NO:97, C is SEQ ID NO:28, D is SEQ ID NO:29, E isSEQ ID NO:30, and F is SEQ ID NO:31, wherein Xaa is alanine, and whereinS1, S2, S3, S4 and S5 are spacers. This invention intends to capture allcombinations of homopolyligands and heteropolyligands without limitationto the examples given above or below. In this description, use of theterm “ligand(s)” encompasses monomeric ligands, polymeric ligands,homopolymeric ligands and/or heteropolymeric ligands.

Monomeric ligands can be categorized into types (FIG. 15). One type ofmonomeric ligand is a polypeptide where at least a portion of thepolypeptide is capable of being recognized by ERK as a substrate orpseudosubstrate (active site blocker). The portion of the polypeptidecapable of recognition is termed the recognition motif. In the presentinvention, recognition motifs can be natural or synthetic. Examples ofrecognition motifs are well known in the art and include, but are notlimited to, naturally occurring ERK substrates and pseudosubstratemotifs (SEQ ID NOS:28-90 and subsequences of SEQ ID NOS:9-27 containinga recognition motif). Another type of monomeric ligand is a polypeptidewhere at least a portion of the polypeptide is capable of associatingwith ERK at a substrate or pseudosubstrate docking site (docking siteblocker). A docking site type of monomeric ligand prevents ERK substratephosphorylation by interfering with substrate association and alignment(SEQ ID NOS:97-108). Yet another type of monomeric ligand is apolypeptide where at least a portion of the polypeptide is capable ofassociating with ERK at ERK's activation site (SEQ ID NOS: 91-96),thereby blocking ERK activation (activation site blocker), therebypreventing ERK from phosphorylating a substrate.

A polymeric ligand comprises two or more monomeric ligands linkedtogether.

A homopolymeric ligand is a polymeric ligand where each of the monomericligands is identical in amino acid sequence, except that aphosphorylatable residue may be substituted or modified in one or moreof the monomeric ligands.

A heteropolymeric ligand is a polymeric ligand where some of themonomeric ligands do not have an identical amino acid sequence.

The ligands of the invention are optionally linked to additionalmolecules or amino acids that provide an epitope tag, a reporter, and/ora cellular localization signal. The cellular localization signal targetsthe ligands to a region of a cell. The epitope tag and/or reporterand/or localization signal may be the same molecule. The epitope tagand/or reporter and/or localization signal may also be differentmolecules.

The invention also encompasses polynucleotides comprising a nucleotidesequence encoding ligands, homopolyligands, and heteropolyligands. Thenucleic acids of the invention are optionally linked to additionalnucleotide sequences encoding polypeptides with additional features,such as an epitope tag, a reporter, and/or a cellular localizationsignal. The polynucleotides are optionally flanked by nucleotidesequences comprising restriction endonuclease sites and othernucleotides needed for restriction endonuclese activity. The flankingsequences optionally provide unique cloning sites within a vector andoptionally provide directionality of subsequence cloning. Further, thenucleic acids of the invention are optionally incorporated into vectorpolynucleotides. The ligands, polyligands, and polynucleotides of thisinvention have utility as research tools and/or therapeutics.

Terms used in the specification and claims are intended to have meaningsconsistent with that known in the art. For example, as used herein, G418is an aminoglycoside antibiotic also known as Geneticin. Resistance toG418 is conferred by the neo gene. HEK293 cells are human embryonickidney 293 cell line. H-RasV12 is a constitutively active mutant form ofRas. NIH3T3 is a mouse fibroblast cell line. Raf stand for Ras-activatedfactor. Ras is a small GTPase or G protein. RNA stands for ribonucleicacid. SiRNA stands for small interfering RNA. Transfection is theintroduction of foreign material (such as DNA) into eukaryotic cells.Transformation is a process of tumorigenesis whereby normal cells becomecancerous and possess phenotypes including but not limited to excessivegrowth, plasticity, chromosome abnormalities, foci formation, cell cycleabnormalities, among others. V-Src is a tyrosine kinase encoded by theviral oncogene isolated from Rous sarcoma virus.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to ligands and polyligands that are ERKmodulators. Various embodiments of ligands and polyligands arerepresented in SEQ ID NOS:1-108. Polyligands are chimeric ligandscomprising two or more monomeric polypeptide ligands. An example of amonomeric ligand is the polypeptide represented by SEQ ID NO:43, whereinXaa is any amino acid. SEQ ID NO:43 is a selected subsequence ofwild-type full length SEQ ID NO:11, wherein the amino acid correspondingto Xaa in the wild-type sequence is a serine or threoninephosphorylatable by ERK. Another example of a monomeric ligand is thepolypeptide represented by SEQ ID NO:99. Another example of a monomericligand is the polypeptide represented by SEQ ID NO:94. Each of SEQ IDNOS:28-108 represents an individual polypeptide ligand in monomericform, wherein Xaa is any amino acid. SEQ ID NOS:28-90 are selectedexamples of subsequences of SEQ ID NOS:9-27, however, other subsequencesof SEQ ID NOS:9-27 containing a recognition motif may also be utilizedas monomeric ligands. Monomeric ligand subsequences of SEQ ID NOS:9-27may be wild-type subsequences. Additionally, monomeric ligandsubsequences of SEQ ID NOS:9-27 may have the ERK phosphorylatable aminoacids replaced by other amino acids. Furthermore, monomeric ligands andpolyligands may have at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or99% sequence identity to a ligand comprising an amino acid sequence inone or more of SEQ ID NOS:28-108. Furthermore, monomeric ligands andpolyligands may have at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%and 99% sequence identity to a subsequence of SEQ ID NOS:9-27.

An example of a homopolyligand is a polypeptide comprising a dimer ormultimer of SEQ ID NO:86, wherein Xaa is any amino acid. Another exampleof a homopolyligand is a polypeptide comprising a dimer or multimer ofSEQ ID NO:95. Another example of a homopolyligand is a polypeptidecomprising a dimer or multimer of SEQ ID NO:106.

An example of a heteropolyligand is a polypeptide comprising SEQ IDNO:108 and one or more of SEQ ID NOS:28-107, wherein Xaa is any aminoacid. There are numerous ways to combine SEQ ID NOS:28-108 intohomopolymeric or heteropolymeric ligands. Furthermore, there arenumerous ways to combine additional subsequences of SEQ ID NOS:9-27 witheach other and with SEQ ID NOS:28-108 to make polymeric ligands.

Polyligands may comprise any two or more of SEQ ID NOS:28-108, whereinXaa is any amino acid. A dimer or multimer of SEQ ID NO:91 is an exampleof a homopolyligand. An example of a heteropolyligand is a polypeptidecomprising SEQ ID NO:28 and one or more of SEQ ID NOS:29-108. There arenumerous ways to combine SEQ ID NOS:28-108 into homopolymeric orheteropolymeric ligands. SEQ ID NOS:28-90 are selected examples ofsubsequences of SEQ ID NOS:9-27, however, additional subsequences,wild-type or mutated, may be utilized to form polyligands. The instantinvention is directed to all possible combinations of homopolyligandsand heteropolyligands without limitation.

SEQ ID NOS:9-27 show proteins that contain at least one serine orthreonine residue phosphorylatable by ERK, the positions of which arerepresented by Xaa. SEQ ID NOS:28-90 are subsequences of SEQ ID NOS:9-27where, again, the locations of the ERK phosphorylatable residues arerepresented by Xaa. In nature, Xaa is, generally speaking, serine orthreonine. In one embodiment of the instant invention, Xaa can be anyamino acid. Ligands where Xaa is serine or threonine can be used as partof a polyligand, however in one embodiment, at least onephosphorylatable serine or threonine is replaced with another aminoacid, such as one of the naturally occurring amino acids including,alanine, aspartate, asparagine, cysteine, glutamate, glutamine,phenylalanine, glycine, histidine, isoleucine, leucine, lysine,methionine, proline, arginine, valine, tryptophan, or tyrosine. The Xaamay also be a non-naturally occurring amino acid. In another embodiment,the ERK phosphorylatable serine(s) or threonine(s) are replaced byalanine. The ligands and polyligands of the invention are designed tomodulate the endogenous effects of one or more isoforms of ERK.

In general, ligand monomers based on natural ERK substrates are built byisolating a putative ERK phosphorylation recognition motif in a ERKsubstrate. Sometimes it is desirable to modify the phosphorylatableresidue to an amino acid other than serine or threonine. Additionalmonomers include the ERK recognition motif as well as amino acidsadjacent and contiguous on either side of the ERK recognition motif.Monomeric ligands may therefore be any length provided the monomerincludes the ERK recognition motif. For example, the monomer maycomprise an ERK recognition motif and at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30-100 or more amino acids adjacent to the recognitionmotif.

For example, in one embodiment, the invention comprises an inhibitor ofERK comprising at least one copy of a peptide selected from the groupconsisting of:

a) a peptide at least 80% identical to a peptide comprising amino acidresidues corresponding to amino acid residues 407-415 of SEQ ID NO:9,wherein the amino acid residue corresponding to amino acid residue 412of SEQ ID NO:9 is an amino acid residue other than serine or threonine;b) a peptide at least 80% identical to a peptide comprising amino acidresidues corresponding to amino acid residues 403-416 of SEQ ID NO:9,wherein the amino acid residue corresponding to amino acid residue 412of SEQ ID NO:9 is an amino acid residue other than serine or threonine;c) a peptide at least 80% identical to a peptide comprising amino acidresidues corresponding to amino acid residues 400-417 of SEQ ID NO:9,wherein the amino acid residue corresponding to amino acid residue 412of SEQ ID NO:9 is an amino acid residue other than serine or threonine;andd) a peptide at least 80% identical to a peptide comprising amino acidresidues corresponding to amino acid residues 399-418 of SEQ ID NO:9,wherein the amino acid residue corresponding to amino acid residue 412of SEQ ID NO:9 is an amino acid residue other than serine or threonine.

As used herein, the terms “correspond(s) to” and “corresponding to,” asthey relate to sequence alignment, are intended to mean enumeratedpositions within a reference protein, e.g., CDC25c (SEQ ID NO:12), andthose positions that align with the positions on the reference protein.Thus, when the amino acid sequence of a subject peptide is aligned withthe amino acid sequence of a reference peptide, e.g., SEQ ID NO:12, theamino acids in the subject peptide sequence that “correspond to” certainenumerated positions of the reference peptide sequence are those thatalign with these positions of the reference peptide sequence, but arenot necessarily in these exact numerical positions of the referencesequence. Methods for aligning sequences for determining correspondingamino acids between sequences are described below.

Additional embodiments of the invention include monomers (as describedabove) based on any putative or real substrate for ERK, such assubstrates identified by SEQ ID NOS:9-27. Furthermore, if the substratehas more than one recognition motif, then more than one monomer may beidentified therein.

Further embodiments of the invention include monomers based on ERKinhibitors, regulators, or binding partners, such as those identified bySEQ ID NOS:91-108 (ERK activation site blockers and ERK substratedocking site blockers) and subsequences thereof.

Another embodiment of the invention is a nucleic acid moleculecomprising a polynucleotide sequence encoding at least one copy of aligand peptide.

Another embodiment of the invention is a nucleic acid molecule whereinthe polynucleotide sequence encodes one or more copies of one or morepeptide ligands.

Another embodiment of the invention is a nucleic acid molecule whereinthe polynucleotide sequence encodes at least a number of copies of thepeptide selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9 or10.

Another embodiment of the invention is a vector comprising a nucleicacid molecule encoding at least one copy of a ligand or polyligand.

Another embodiment of the invention is a recombinant host cellcomprising a vector comprising a nucleic acid molecule encoding at leastone copy of a ligand or polyligand.

Another embodiment of the invention is a method of inhibiting ERK in acell comprising transfecting a vector comprising a nucleic acid moleculeencoding at least one copy of a ligand or polyligand into a host celland culturing the transfected host cell under conditions suitable toproduce at least one copy of the ligand or polyligand.

The invention also relates to modified inhibitors that are at leastabout 80%, 85%, 90% 95%, 96%, 97%, 98% or 99% identical to a referenceinhibitor. A “modified inhibitor” is used to mean a peptide that can becreated by addition, deletion or substitution of one or more amino acidsin the primary structure (amino acid sequence) of a inhibitor protein orpolypeptide. A “modified recognition motif” is a naturally occurring ERKrecognition motif that has been modified by addition, deletion, orsubstitution of one or more amino acids in the primary structure (aminoacid sequence) of the motif. For example, a modified ERK recognitionmotif may be a motif where the phosphorylatable amino acid has beenmodified to a non-phosphorylatable amino acid. The terms “protein” and“polypeptide” are used interchangeably herein. The reference inhibitoris not necessarily a wild-type protein or a portion thereof. Thus, thereference inhibitor may be a protein or peptide whose sequence waspreviously modified over a wild-type protein. The reference inhibitormay or may not be the wild-type protein from a particular organism.

A polypeptide having an amino acid sequence at least, for example, about95% “identical” to a reference an amino acid sequence is understood tomean that the amino acid sequence of the polypeptide is identical to thereference sequence except that the amino acid sequence may include up toabout five modifications per each 100 amino acids of the reference aminoacid sequence encoding the reference peptide. In other words, to obtaina peptide having an amino acid sequence at least about 95% identical toa reference amino acid sequence, up to about 5% of the amino acidresidues of the reference sequence may be deleted or substituted withanother amino acid or a number of amino acids up to about 5% of thetotal amino acids in the reference sequence may be inserted into thereference sequence. These modifications of the reference sequence mayoccur at the N-terminus or C-terminus positions of the reference aminoacid sequence or anywhere between those terminal positions, interspersedeither individually among amino acids in the reference sequence or inone or more contiguous groups within the reference sequence.

As used herein, “identity” is a measure of the identity of nucleotidesequences or amino acid sequences compared to a reference nucleotide oramino acid sequence. In general, the sequences are aligned so that thehighest order match is obtained. “Identity” per se has an art-recognizedmeaning and can be calculated using published techniques. (See, e.g.,Computational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York (1988); Biocomputing: Informatics And Genome Projects,Smith, D. W., ed., Academic Press, New York (1993); Computer Analysis ofSequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., HumanaPress, New Jersey (1994); von Heinje, G., Sequence Analysis In MolecularBiology, Academic Press (1987); and Sequence Analysis Primer, Gribskov,M. and Devereux, J., eds., M Stockton Press, New York (1991)). Whilethere exist several methods to measure identity between twopolynucleotide or polypeptide sequences, the term “identity” is wellknown to skilled artisans (Carillo, H. & Lipton, D., Siam J Applied Math48:1073 (1988)). Methods commonly employed to determine identity orsimilarity between two sequences include, but are not limited to, thosedisclosed in Guide to Huge Computers, Martin J. Bishop, ed., AcademicPress, San Diego (1994) and Carillo, H. & Lipton, D., Siam J AppliedMath 48:1073 (1988). Computer programs may also contain methods andalgorithms that calculate identity and similarity. Examples of computerprogram methods to determine identity and similarity between twosequences include, but are not limited to, GCG program package(Devereux, J., et al., Nucleic Acids Research 12(i):387 (1984)), BLASTP,ExPASy, BLASTN, FASTA (Atschul, S. F., et al., J Molec Biol 215:403(1990)) and FASTDB. Examples of methods to determine identity andsimilarity are discussed in Michaels, G. and Garian, R., CurrentProtocols in Protein Science, Vol 1, John Wiley & Sons, Inc. (2000),which is incorporated by reference. In one embodiment of the presentinvention, the algorithm used to determine identity between two or morepolypeptides is BLASTP.

In another embodiment of the present invention, the algorithm used todetermine identity between two or more polypeptides is FASTDB, which isbased upon the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245(1990), incorporated by reference). In a FASTDB sequence alignment, thequery and subject sequences are amino sequences. The result of sequencealignment is in percent identity. Parameters that may be used in aFASTDB alignment of amino acid sequences to calculate percent identityinclude, but are not limited to: Matrix=PAM, k-tuple=2, MismatchPenalty=1, Joining Penalty=20, Randomization Group Length=0, CutoffScore=1, Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or thelength of the subject amino sequence, whichever is shorter.

If the subject sequence is shorter or longer than the query sequencebecause of N-terminus or C-terminus additions or deletions, not becauseof internal additions or deletions, a manual correction can be made,because the FASTDB program does not account for N-terminus andC-terminus truncations or additions of the subject sequence whencalculating percent identity. For subject sequences truncated at bothends, relative to the query sequence, the percent identity is correctedby calculating the number of amino acids of the query sequence that areN- and C-terminus to the reference sequence that are notmatched/aligned, as a percent of the total amino acids of the querysequence. The results of the FASTDB sequence alignment determinematching/alignment. The alignment percentage is then subtracted from thepercent identity, calculated by the above FASTDB program using thespecified parameters, to arrive at a final percent identity score. Thiscorrected score can be used for the purposes of determining howalignments “correspond” to each other, as well as percentage identity.Residues of the query (subject) sequences or the reference sequence thatextend past the N- or C-termini of the reference or subject sequence,respectively, may be considered for the purposes of manually adjustingthe percent identity score. That is, residues that are notmatched/aligned with the N- or C-termini of the comparison sequence maybe counted when manually adjusting the percent identity score oralignment numbering.

For example, a 90 amino acid residue subject sequence is aligned with a100 residue reference sequence to determine percent identity. Thedeletion occurs at the N-terminus of the subject sequence and therefore,the FASTDB alignment does not show a match/alignment of the first 10residues at the N-terminus. The 10 unpaired residues represent 10% ofthe sequence (number of residues at the N- and C-termini notmatched/total number of residues in the query sequence) so 10% issubtracted from the percent identity score calculated by the FASTDBprogram. If the remaining 90 residues were perfectly matched the finalpercent identity would be 90%. In another example, a 90 residue subjectsequence is compared with a 100 reference sequence. This time thedeletions are internal deletions so there are no residues at the N- orC-termini of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected.

The polyligands of the invention optionally comprise spacer amino acidsbefore, after, or between monomers. The length and composition of thespacer may vary. An example of a spacer is glycine, alanine,polyglycine, or polyalanine. Specific examples of spacers used betweenmonomers in SEQ ID NO:5 are the four amino acid spacers AAAA, GAGA,GGGG, AGAG, AAGG, GGAA, and the six amino acid spacer AGPGAEF. In theinstance of SEQ ID NO:5, the proline-containing spacer is intended tobreak an alpha helical secondary structure. Spacer amino acids may beany amino acid and are not limited to alanine, glycine and proline. Theinstant invention is directed to all combinations of homopolyligands andheteropolyligands, with or without spacers, and without limitation tothe examples given above or below.

The ligands and polyligands of the invention are optionally linked toadditional molecules or amino acids that provide an epitope tag, areporter, and/or localize the ligand to a region of a cell (See FIGS.5A-5G, FIGS. 6A-6G, FIGS. 7A-7G, and FIGS. 8A-8G). Non-limiting examplesof epitope tags are FLAG™ (Kodak; Rochester, N.Y.), HA (hemagluttinin),c-Myc and His6. Non-limiting examples of reporters are alkalinephosphatase, galactosidase, peroxidase, luciferase and green fluorescentprotein (GFP). Non-limiting examples of cellular localizations aresarcoplamic reticulum, endoplasmic reticulum, mitochondria, golgiapparatus, nucleus, plasma membrane, apical membrane, and basolateralmembrane. The epitopes, reporters and localization signals are given byway of example and without limitation. The epitope tag, reporter and/orlocalization signal may be the same molecule. The epitope tag, reporterand/or localization signal may also be different molecules.

Ligands and polyligands and optional amino acids linked thereto can besynthesized chemically or recombinantly using techniques known in theart. Chemical synthesis techniques include but are not limited topeptide synthesis which is often performed using an automated peptidesynthesizer. Pepetides can also be synthesized utilizing non-automatedpeptide synthesis methods known in the art. Recombinant techniquesinclude insertion of ligand-encoding nucleic acids into expressionvectors, wherein nucleic acid expression products are synthesized usingcellular factors and processes.

Linkage of a cellular localization signal, epitope tag, or reporter to aligand or polyligand can include covalent or enzymatic linkage to theligand. When the localization signal comprises material other than apolypeptide, such as a lipid or carbohydrate, a chemical reaction tolink molecules may be utilized. Additionally, non-standard amino acidsand amino acids modified with lipids, carbohydrates, phosphate or othermolecules may be used as precursors to peptide synthesis. The ligands ofthe invention have therapeutic utility with or without localizationsignals. However, ligands linked to localization signals have utility assubcellular tools or therapeutics. For example, ligands depictedgenerically in FIGS. 7A-7G represent ligands with utility as subcellulartools or therapeutics. ERK ligand-containing gene constructs are alsodelivered via gene therapy. FIGS. 10B and 10C depict embodiments of genetherapy vectors for delivering and controlling polypeptide expression invivo. Polynucleotide sequences linked to the gene construct in FIGS. 10Band 10C include genome integration domains to facilitate integration ofthe transgene into a viral genome and/or host genome.

FIG. 10A shows a vector containing an ERK ligand gene construct, whereinthe ligand gene construct is releasable from the vector as a unit usefulfor generating transgenic animals. For example, the ligand geneconstruct, or transgene, is released from the vector backbone byrestriction endonuclease digestion. The released transgene is theninjected into pronuclei of fertilized mouse eggs; or the transgene isused to transform embryonic stem cells. The vector containing a ligandgene construct of FIG. 10A is also useful for transient transfection ofthe trangene, wherein the promoter and codons of the transgene areoptimized for the host organism. The vector containing a ligand geneconstruct of FIG. 10A is also useful for recombinant expression ofpolypeptides in fermentable organisms adaptable for small or large scaleproduction, wherein the promoter and codons of the transgene areoptimized for the fermentation host organism.

FIG. 10D shows a vector containing an ERK ligand gene construct usefulfor generating stable cell lines.

The invention also encompasses polynucleotides comprising nucleotidesequences encoding ligands, homopolyligands, and heteropolyligands. Thepolynucleotides of the invention are optionally linked to additionalnucleotide sequences encoding epitopes, reporters and/or localizationsignals. Further, the nucleic acids of the invention are optionallyincorporated into vector polynucleotides. The polynucleotides areoptionally flanked by nucleotide sequences comprising restrictionendonuclease sites and other nucleotides needed for restrictionendonuclese activity. The flanking sequences optionally provide cloningsites within a vector. The restriction sites can include, but are notlimited to, any of the commonly used sites in most commerciallyavailable cloning vectors. Examples of such sites are those recognizedby BamHI, ClaI, EcoRI, EcoRV, SpeI, Anil, NdeI, NheI, XbaI, XhoI, SphI,NaeI, SexAI, HindIII, HpaI, and PstI restriction endonucleases. Sitesfor cleavage by other restriction enzymes, including homingendonucleases, are also used for this purpose. The polynucleotideflanking sequences also optionally provide directionality of subsequencecloning. It is preferred that 5′ and 3′ restriction endonuclease sitesdiffer from each other so that double-stranded DNA can be directionallycloned into corresponding complementary sites of a cloning vector.

Ligands and polyligands with or without localization signals, epitopesor reporters are alternatively synthesized by recombinant techniques.Polynucleotide expression constructs are made containing desiredcomponents and inserted into an expression vector. The expression vectoris then transfected into cells and the polypeptide products areexpressed and isolated. Ligands made according to recombinant DNAtechniques have utility as research tools and/or therapeutics.

The following is an example of how polynucleotides encoding ligands andpolyligands are produced. Complimentary oligonucleotides encoding theligands and flanking sequences are synthesized and annealed. Theresulting double-stranded DNA molecule is inserted into a cloning vectorusing techniques known in the art. When the ligands and polyligands areplaced in-frame adjacent to sequences within a transgenic gene constructthat is translated into a protein product, they form part of a fusionprotein when expressed in cells or transgenic animals.

Another embodiment of the invention relates to selective control oftransgene expression in a desired cell or organism. The promotor portionof the recombinant gene can be a constitutive promotor, anon-constitutive promotor, a tissue-specific promotor (constitutive ornon-constitutive) or a selectively controlled promotor. Differentselectively controlled promotors are controlled by different mechanisms.For example, RHEOSWITCH is an inducible promotor system available fromNew England Biolabs (Ipswich, Mass.). Temperature sensitive promotorscan also be used to increase or decrease gene expression. An embodimentof the invention comprises a ligand or polyligand gene construct whoseexpression is controlled by an inducible promotor. In one embodiment,the inducible promotor is tetracycline controllable.

Polyligands are modular in nature. An aspect of the instant invention isthe combinatorial modularity of the disclosed polyligands. Anotheraspect of the invention are methods of making these modular polyligandseasily and conveniently. In this regard, an embodiment of the inventioncomprises methods of modular subsequence cloning of genetic expressioncomponents. When the ligands, homopolyligands, heteropolyligands andoptional amino acid expression components are synthesized recombinantly,one can consider each clonable element as a module. For speed andconvenience of cloning, it is desirable to make modular elements thatare compatible at cohesive ends and are easy to insert and clonesequentially. This is accomplished by exploiting the natural propertiesof restriction endonuclease site recognition and cleavage. One aspect ofthe invention encompasses module flanking sequences that, at one end ofthe module, are utilized for restriction enzyme digestion once, and atthe other end, utilized for restriction enzyme digestion as many timesas desired. In other words, a restriction site at one end of the moduleis utilized and destroyed in order to effect sequential cloning ofmodular elements. An example of restriction sites flanking a codingregion module are sequences recognized by the restriction enzymes NgoMIV and Cla I; or Xma I and Cla I. Cutting a first circular DNA with NgoMIV and Cla I to yield linear DNA with a 5′ NgoM IV overhang and a 3′ ClaI overhang; and cutting a second circular DNA with Xma I and Cla I toyield linear DNA with a 5′ Cla I overhang and a 3′ Xma I overhanggenerates first and second DNA fragments with compatible cohesive ends.When these first and second DNA fragments are mixed together, annealed,and ligated to form a third circular DNA fragment, the NgoM IV site thatwas in the first DNA and the Xma I site that was in the second DNA aredestroyed in the third circular DNA. Now this vestigial region of DNA isprotected from further Xma I or NgoM IV digestion, but flankingsequences remaining in the third circular DNA still contain intact 5′NgoM IV and 3′ Cla I sites. This process can be repeated numerous timesto achieve directional, sequential, modular cloning events. Restrictionsites recognized by NgoM IV, Xma I, and Cla I endonucleases represent agroup of sites that permit sequential cloning when used as flankingsequences.

Another way to assemble coding region modules directionally andsequentially employs linear DNA in addition to circular DNA. Forexample, like the sequential cloning process described above,restriction sites flanking a coding region module are sequencesrecognized by the restriction enzymes NgoM IV and Cla I; or Xma I andCla I. A first circular DNA is cut with NgoM IV and Cla I to yieldlinear DNA with a 5′ NgoM IV overhang and a 3′ Cla I overhang. A secondlinear double-stranded DNA is generated by PCR amplification or bysynthesizing and annealing complimentary oligonucleotides. The secondlinear DNA has 5′ Cla I overhang and a 3′ Xma I overhang, which arecompatible cohesive ends with the first DNA linearized. When these firstand second DNA fragments are mixed together, annealed, and ligated toform a third circular DNA fragment, the NgoM IV site that was in thefirst DNA and the Xma I site that was in the second DNA are destroyed inthe third circular DNA. Flanking sequences remaining in the thirdcircular DNA still contain intact 5′ NgoM IV and 3′ Cla I sites. Thisprocess can be repeated numerous times to achieve directional,sequential, modular cloning events. Restriction sites recognized by NgoMIV, Xma I, and Cla I endonucleases represent a group of sites thatpermit sequential cloning when used as flanking sequences. This processis depicted in FIG. 11.

One of ordinary skill in the art recognizes that other restriction sitegroups can accomplish sequential, directional cloning as describedherein. Preferred criteria for restriction endonuclease selection areselecting a pair of endonucleases that generate compatible cohesive endsbut whose sites are destroyed upon ligation with each other. Anothercriteria is to select a third endonuclease site that does not generatesticky ends compatible with either of the first two. When such criteriaare utilized as a system for sequential, directional cloning, ligands,polyligands and other coding regions or expression components can becombinatorially assembled as desired. The same sequential process can beutilized for epitope, reporter, and/or localization signals.

Polyligands and methods of making polyligands that modulate ERK activityare disclosed. Therapeutics include delivery of purified ligand orpolyligand with or without a localization signal to a cell.Alternatively, ligands and polyligands with or without a localizationsignals are delivered via adenovirus, lentivirus, adeno-associatedvirus, or other viral constructs that express protein product in a cell.

Methods

Assays. Ligands of the invention are assayed for kinase modulatingactivity using one or more of the following methods.

Method 1. A biochemical assay is performed employingcommercially-obtained kinase, commercially-obtained substrate,commercially-obtained kinase inhibitor (control), and semi-purifiedinhibitor ligand of the invention (decoy ligand). Decoy ligands arelinked to an epitope tag at one end of the polypeptide for purificationand/or immobilization, for example, on a microtiter plate. The taggeddecoy ligand is made using an in vitro transcription/translation systemsuch as a reticulocyte lysate system well known in the art. A vectorpolynucleotide comprising a promotor, such as T7 and/or T3 and/or SP6promotor, a decoy ligand coding sequence, and an epitope tag codingsequence is employed to synthesize the tagged decoy ligand in an invitro transcription/translation system. In vitrotranscription/translation protocols are disclosed in reference manualssuch as: Current Protocols in Molecular Biology (eds. Ausubel et al.,Wiley, 2004 edition.) and Molecular Cloning: A Laboratory Manual(Sambrook and Russell (Cold Spring Harbor Laboratory Press, 2001, thirdedition). Immunoreagent-containing methods such as western blots,elisas, and immunoprecipitations are performed as described in: UsingAntibodies: A Laboratory Manual (Harlow and Lane Cold Spring HarborLaboratory Press, 1999).

Specifically, tagged decoy ligand synthesized using an in vitrotranscription/translation system is semi-purified and added to amicrotiter plate containing kinase enzyme and substrate immobilized byan anti-substrate specific antibody. Microtiter plates are rinsed tosubstantially remove non-immobilized components. Kinase activity is adirect measure of the phosphorylation of substrate by kinase employing aphospho-substrate specific secondary antibody conjugated to horseradishperoxidase (HRP) followed by the addition of3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution. The catalysisof TMB by HRP results in a blue color that changes to yellow uponaddition of phosphoric or sulfuric acid with a maximum absorbance at 450nm. The Control experiments include absence of kinase enzyme, and/orabsence of decoy ligand, and/or presence/absence of known kinaseinhibitors. A known kinase inhibitor useful in the assay isstaurosporine.

Method 2. A similar assay is performed employing the same reagents asabove but the substrate is biotinylated and immobilized by binding to astreptavidin-coated plate.

Method 3. A biochemical assay is performed employingcommercially-obtained kinase, commercially-obtained substrate,commercially-obtained kinase inhibitor (control), and semi-purifiedinhibitor ligand of the invention (decoy ligand) in a microtiter plate.A luminescent-based detection system, such as Promega's Kinase-Glo, isthen added to inversely measure kinase activity.

Specifically, tagged decoy ligand synthesized using an in vitrotranscription/translation system is semi-purified and added to amicrotiter plate containing kinase enzyme and substrate. After thekinase assay is performed, luciferase and luciferin are added to thereaction. Luciferase utilizes any remaining ATP not used by the kinaseto catalyze luciferin. The luciferase reaction results in the productionof light which is inversely related to kinase activity. Controlexperiments include absence of kinase enzyme, and/or absence of decoyligand, and/or presence/absence of known kinase inhibitors. A knownkinase inhibitor useful in the assay is staurosporine.

Method 4. A similar cell-based assay is performed employing samereagents as above, but synthesizing the decoy ligand in a mammalian cellsystem instead of an in vitro transcription/translation system. Decoyligands are linked to an epitope tag at one end of the polypeptide forimmobilzation and/or for purification and/or for identification in awestern blot. Optionally, tagged decoy ligands are also linked to acellular localization signal for phenotypic comparison of pan-cellularand localized kinase modulation. A vector polynucleotide comprising aconstitutive promotor, such as the CMV promotor, a decoy ligand codingsequence, an epitope tag coding sequence, and optionally a localizationsignal coding sequence is employed to express the decoy ligand in cells.Transfection and expression protocols are disclosed in reference manualssuch as: Current Protocols in Molecular Biology (eds. Ausubel et al.,Wiley, 2004 edition.) and Molecular Cloning: A Laboratory Manual(Sambrook and Russell (Cold Spring Harbor Laboratory Press, 2001, thirdedition). Western Blots and immunoreagent-containing methods areperformed as described in: Using Antibodies: A Laboratory Manual (Harlowand Lane Cold Spring Harbor Laboratory Press, 1999).

EXAMPLES Example 1

A polypeptide comprising a heteropolyligand, an endoplasmic reticulumcellular localization signal, and a His6 epitope is synthesized.Examples of such polypeptides are generically represented by FIGS. 8A,8B, 8D, 8E and 8F. The polypeptide is synthesized on an automatedpeptide synthesizer or is recombinantly expressed and purified. Purifiedpolypeptide is solubilized in media and added to cells. The polypeptideis endocytosed by the cells, and transported to the endoplasmicreticulum. Verification is performed by immunohistochemical stainingusing an anti-His6 antibody.

Example 2

A transgene is constructed using a human cytomegalovirus (CMV) promoterto direct expression of a fusion protein comprising SEQ ID NO:96, SEQ IDNO:99, SEQ ID NO:89, wherein Xaa is alanine (POLYLIGAND), greenfluorescent protein (REPORTER), and a plasma membrane localizationsignal (LOCALIZATION SIGNAL). Such a transgene is genericallyrepresented by FIG. 9C. The transgene is transfected into cells fortransient expression. Verification of expression and location isperformed by visualization of green fluorescent protein (GFP) byconfocal microscopy.

Example 3

A transgene construct is built to produce a protein product withexpression driven by a tissue-specific promoter. The transgene comprisesa synthetic gene expression unit engineered to encode three domains.Each of these three domains is synthesized as a pair of complimentarypolynucleotides that are annealed in solution, ligated and inserted intoa vector. Starting at the amino-terminus, the three domains in theexpression unit are nucleotide sequences that encode an ERK ligand, aFLAG™ epitope, and a nuclear localization signal. The ERK ligand is amonomeric ligand, homopolymeric ligand or heteropolymeric ligand asdescribed herein. Nucleotide sequences encoding a FLAG™ epitope areplaced downstream of nucleotide sequences encoding the ERK ligand.Finally, nucleotide sequences encoding the localization signal areplaced downstream of those encoding the FLAG™ epitope. The assembledgene expression unit is subsequently subcloned into an expressionvector, such as that shown in FIG. 10A, and used to transientlytransfect cells. Verification is performed by immunohistochemicalstaining using an anti-FLAG™ antibody.

Example 4

Modulation of ERK cellular function by subcellularly localized ERKpolyligand is illustrated. A transgene construct containing nucleicacids that encode a polyligand fusion protein, epitope, and endoplasmicreticulum localization signal is made. The expression unit containsnucleotides that encode SEQ ID NO:1 (POLYLIGAND), a c-Myc epitope(EPITOPE), and an endoplasmic reticulum localization signal(LOCALIZATION SIGNAL). This expression unit is subsequently subclonedinto a vector between a EF1alpha promoter and an SV40 polyadenylationsignal (depicted in FIG. 12). The completed transgene-containingexpression vector is then used to transfect cells. Inhibition of ERKactivity is demonstrated by measuring phosphorylation of endogenoussubstrates against controls (see FIG. 14).

Example 5

Ligand function and localization is demonstrated in vivo by making atransgene construct used to generate mice expressing a ligand fusionprotein targeted to the endoplasmic reticulum. The transgene constructis shown generically in FIG. 10B. The expression unit containsnucleotides that encode a tetramer of SEQ ID NO:65, a hemagluttininepitope, and a nuclear localization signal. This expression unit issubsequently subcloned into a vector between nucleotide sequencesincluding an inducible promoter and an SV40 polyadenylation signal. Thecompleted transgene is then injected into pronuclei of fertilized mouseoocytes. The resultant pups are screened for the presence of thetransgene by PCR. Transgenic founder mice are bred with wild-type mice.Heterozygous transgenic animals from at least the third generation areused for the following tests, with their non-transgenic littermatesserving as controls.

Test 1: Southern blotting analysis is performed to determine the copynumber. Southern blots are hybridized with a radio-labeled probegenerated from a fragment of the transgene. The probe detects bandscontaining DNA from transgenic mice, but does not detect bandscontaining DNA from non-transgenic mice. Intensities of the transgenicmice bands are measured and compared with the transgene plasmid controlbands to estimate copy number. This demonstrates that mice in Example 5harbor the transgene in their genomes.

Test 2: Tissue homogenates are prepared for Western blot analysis. Thisexperiment demonstrates the transgene is expressed in tissues oftransgenic mice because hemagluttinin epitope is detected in transgenichomogenates but not in non-transgenic homogenates.

Test 3: Function is assessed by phenotypic observation or analysisagainst controls.

These examples demonstrate delivery of ligands to a localized region ofa cell for therapeutic or experimental purposes. The purifiedpolypeptide ligands can be formulated for oral or parenteraladministration, topical administration, or in tablet, capsule, or liquidform, intranasal or inhaled aerosol, subcutaneous, intramuscular,intraperitoneal, or other injection; intravenous instillation; or anyother routes of administration. Furthermore, the nucleotide sequencesencoding the ligands permit incorporation into a vector designed todeliver and express a gene product in a cell. Such vectors includeplasmids, cosmids, artificial chromosomes, and modified viruses.Delivery to eukaryotic cells can be accomplished in vivo or ex vivo. Exvivo delivery methods include isolation of the intended recipient'scells or donor cells and delivery of the vector to those cells, followedby treatment of the recipient with the cells.

Results

Results show that the ERK polyligands of the invention (decoys)localized to the appropriate subcellular compartments and inhibited ERKactivity at those locations. Localized inhibition caused distinctfunctional changes in the treated cells, including inhibition ofoncogene-induced cell proliferation and transformation. Furthermore,depending on the source of the activation signal for the ERK pathway,the specific subcellular site of inhibition (endoplasmic reticulum orplasma membrane) had a differentiating effect on transformationphenotype of the cells. In contrast, inhibition of ERK by siRNA or asmall molecule inhibitor did not reveal this functional difference.

Fluorescence microscopy of the ligand of SEQ ID NO:1 is shown localizedto the nucleus (FIG. 16), cytoplasm (FIG. 17), and endoplasmic reticulum(FIG. 13A). The localized ERK ligands were detected by immunostainingfor the c-myc epitope tag. FIG. 14 shows ERK activity localized tospecific compartments with localized Ras overexpression and SEQ ID NO:1expressed pancellularly (lanes 8-9) or targeted to the endoplasmicreticulum (lanes 5-7) or plasma membrane (lanes 2-4). The result waslocation-selective inhibition of ERK activity at the endoplasmicreticulum as measured by phosphorylation of the ERK substrate myelinbasic protein (MBP).

Additionally, experiments where inhibition of ERK signaling using ERKligands of the invention was compared to siRNAs and a small moleculeinhibitor. The commercial siRNAs were designed for target specificity toeither the ERK1 (sc-29307) or ERK2 (sc-35335) isoforms (Santa CruzBiotechnology, Inc.). The small molecule inhibitor, UO126(1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio) butadiene), inhibitsERK signaling through its upstream effector, MEK, and ERK is the onlyknown substrate for MEK. Assays performed were phenotypic assays. Theeffects of inhibiting ERK activity were measured by looking atfunctional properties of the cells associated with the MAPK signalingpathway, such as, cell proliferation and transformation. The MAPKsignaling cascade in these experiments is initiated by transfection ofthe cells with a vector containing a constitutively activeproto-oncogene, either H-RasV12 or v-Src, which eventually causes thecells to acquire enhanced growth rate (colony formation) and celltransformation rate (foci formation). Inhibition of ERK activity in thiscascade will result in reduced rates of proliferation or transformationas measured by numbers of G418-resistant colonies or foci.

Data for colony formation inhibition with the various treatments ispresented in FIG. 18. UO126 is very effective at inhibiting the pathwaydue to its high potency for MEK (˜70 nM) and its stability. Furthermore,the siRNAs targeted against ERK1 or ERK2 show isoform specificity as tothe effects on proliferation. This is consistent with a recent reportthat showed interplay between ERK1 and ERK2 in regulating Ras-mediatedsignaling, wherein ERK2 has a positive role in controlling cellproliferation and ERK1 can affect signal output by counteracting ERK2activity (Vantaggiato et al. 2006 J. of Biol. 5:14). The two ERK ligands(SEQ ID NO:1 and SEQ ID NO:5, both fused to c-Myc and FLAG tags) used inthis experiment are not targeted to a specific subcellular location butare expressed throughout the cell under the control of a constitutivepromoter. The ERK ligands, as described herein, are designed withmultiple domains (usually mutated substrates) believed capable ofcompeting with the normal endogenous ERK substrates. Thus, unlikesiRNAs, which can have RNA sequence specificity for each of the twoisoforms of ERK, the ERK decoy ligands may bind to both ERK1 and ERK2proteins, which may result in only partial inhibition of cellproliferation. Possible reasons for partial inhibition may include atitration effect whereby some of the decoy ligand is “trapped” by ERK1and unavailable to inhibit ERK2. Partial inhibition may also be due tothe inhibition of the ERK1, possibly antagonizing or mitigating theinhibitory effects on ERK2.

Based on the similar effectiveness of SEQ ID NO:1 and SEQ ID NO:5 ininhibiting Ras-mediated cell proliferation, a similar experiment wasconducted with SEQ ID NO:1 localized to the nucleus (NLS), the cytoplasm(NXP, nuclear exclusion), the endoplasmic reticulum (ER), and the plasmamembrane (PLA). In all cases, location-specific SEQ ID NO:1 causes someinhibition of cell proliferation, with the greatest degree of inhibitionarising when the decoy is localized to the plasma membrane (FIG. 19).ER-localized inhibition was also significant, consistent with theresults previously reported using dominant negative location-targetedRas inhibitors (Matallanas et al. (2006) Mol. Cell. Biol. 26: 100-116).SEQ ID NO:1 targeted to the nucleus and cytoplasm gives slightinhibition of proliferation.

Next, the effect of ERK inhibition on cell transformation wasinvestigated using two means of initiating signaling cascades that leadto this biological property (FIG. 20). The first method is theconstitutively active Ras mutant used herein above. The second is aconstitutively active nonreceptor tyrosine kinase v-Src mutant(pp60v-src) that also leads to cell transformation, potentially bymultiple signaling pathways including the Ras-Raf-MEK-ERK pathway. Asshown in FIG. 20, the pancellular decoys (SEQ ID NO:1 and SEQ ID NO:5),ERK2 siRNA, and localized decoy (SEQ ID NO:1 fused to localizationsignals indicated) all inhibited cell transformation. Treatment ofH-RasV 12 transformed cells with ER-localized and PLA-localized SEQ IDNO:1 inhibited cell transformation rates by ˜50%, similar to resultsobtained with the pancellularly expressed SEQ ID NO:1 and SEQ ID NO:5.However, when transformation was initiated with v-Src, there was adifference in the inhibition specificity arising from use of SEQ ID NO:1localized to the ER and PLA. The ER-localized SEQ ID NO:1 caused littleto no inhibition relative to the untreated control, while thePLA-localized SEQ ID NO:1 caused ˜60% decrease in transformation. Thatis, ER-localized SEQ ID NO:1 has a significant effect on transformationinduced by H-RasV12 but little to no effect when transformation isinduced by v-Src. In contrast, inhibition of ERK by siRNA was identicalfor both the H-Ras and v-Src pathways. Thus, siRNA does notdifferentiate the effects on transformation induced by distinctoncogenes H-Ras and v-Src.

Disclosed are ligands and polyligands that modulate ERK activity andmethods of making and using these ligands. The ligands and polyligandsare synthesized chemically or recombinantly and are utilized as researchtools or as therapeutics. The invention includes linking the ligands andpolyligands to cellular localization signals for subcellulartherapeutics.

1.-18. (canceled)
 19. An isolated polynucleotide comprising a nucleotidesequence encoding a polypeptide polyligand comprising two or morepolypeptide that are at least 80% identical to polypeptides selectedfrom SEQ ID NOS: 28, 32, 36, 48, 49, 74, 75, 91, 93-96 and 102, whereinXaa is any amino acid, and wherein the polypeptide polyligand inhibitsthe kinase activity of extracellular-signal-regulated kinase (ERK). 20.The isolated polynucleotide of claim 19, wherein at least one amino aciddesignated as Xaa is an amino acid other than serine or threonine. 21.The isolated polynucleotide of claim 19, wherein said polypeptidepolyligand is a heteropolyligand.
 22. The isolated polynucleotide ofclaim 19, wherein said polypeptide polyligand is a homopolyligand. 23.The isolated polynucleotide of claim 19, wherein said polypeptidepolyligand is linked to one or more of a localization signal, an epitopetag, or a reporter.
 24. A vector comprising the isolated polynucleotideof claim
 19. 25. A host cell comprising the vector of claim
 24. 26. Theisolated polynucleotide of claim 19, operably linked to a promoter. 27.The isolated polynucleotide of claim 26, wherein said promoter is aninducible promoter.
 28. The isolated polynucleotide of claim 19, whereinsaid two or more polypeptides are at least 85% identical to polypeptidesselected from SEQ ID NOS: 28, 32, 36, 48, 49, 74, 75, 91, 93-96 and 102.29. The isolated polynucleotide of claim 19, wherein said two or morepolypeptides are at least 90% identical to polypeptides selected fromSEQ ID NOS: 28, 32, 36, 48, 49, 74, 75, 91, 93-96 and
 102. 30. Theisolated polynucleotide of claim 19, wherein said two or morepolypeptides are at least 95% identical to polypeptides selected fromSEQ ID NOS: 28, 32, 36, 48, 49, 74, 75, 91, 93-96 and
 102. 31. Theisolated polynucleotide of claim 19, wherein said two or morepolypeptides are at least 96% identical to polypeptides selected fromSEQ ID NOS: 28, 32, 36, 48, 49, 74, 75, 91, 93-96 and
 102. 32. Theisolated polynucleotide of claim 19, wherein said two or morepolypeptides are at least 97% identical to polypeptides selected fromSEQ ID NOS: 28, 32, 36, 48, 49, 74, 75, 91, 93-96 and
 102. 33. Theisolated polynucleotide of claim 19, wherein said two or morepolypeptides are at least 98% identical to polypeptides selected fromSEQ ID NOS: 28, 32, 36, 48, 49, 74, 75, 91, 93-96 and
 102. 34. Theisolated polynucleotide of claim 19 wherein said two or morepolypeptides are at least 99% identical to polypeptides selected fromSEQ ID NOS: 28, 32, 36, 48, 49, 74, 75, 91, 93-96 and
 102. 35. Theisolated polynucleotide of claim 19, wherein said two or morepolypeptides are selected from SEQ ID NOS: 28, 32, 36, 48, 49, 74, 75,91, 93-96 and
 102. 36. A method of inhibiting ERK in a cell, said methodcomprising (a) transfecting the vector of claim 24 into a host cell, and(b) culturing the transfected host cell under conditions suitable toproduce at least one copy of the polypeptide polyligand.